The present invention relates to a sensor and a method of determining cholesterol included in low density lipoprotein in a sample like blood, serum, or plasma.
The concentration of cholesterol in low density lipoprotein has been noted as an important parameter in diagnosis of hypercholesterolemia.
The prior art method determines cholesterol in the low density lipoprotein by differential ultracentrifugation. This method, however, requires special equipment and disadvantageously takes a long time for measurement.
A typical method of determination without using the ultracentrifugation respectively measures the total concentration of cholesterol in a sample, the concentration of cholesterol in high density lipoprotein, and the concentration of triglyceride and calculates the concentration of cholesterol in the low density lipoprotein according to the Friedewald""s equation. In the case where the sample contains a high concentration of triglyceride, however, this method has rather low degrees of confidence in reproducibility and accuracy.
Some methods of determining cholesterol in the low density lipoprotein regardless of the concentration of triglyceride have also been proposed.
The first proposed method oxidizes only cholesterol in the low density lipoprotein with an enzyme in the presence of a-cyclodextrin sulfate, dextran sulfate, magnesium ion, and polyoxyethylene-polyoxypropylene block copolyether and determines the concentration of cholesterol in the low density lipoprotein based on the degree of color development of a coloring matter (see Clinical Chemistry, Vol. 44, p522 (1998)). This method uses xcex1-cyclodextrin sulfate, dextran sulfate, and magnesium ion to reduce the reactivity of the enzyme with cholesterol in chylomicron and cholesterol in the low density lipoprotein included in the sample, while using polyoxyethylene-polyoxypropylene block copolyether to reduce the reactivity of the enzyme with cholesterol in the high density lipoprotein in the sample.
The second proposed method oxidizes only cholesterol in the low density lipoprotein with an enzyme in the presence of an amphoteric surface active agent and aliphatic amines having either of carboxyl group and sulfonate group and determines the concentration of cholesterol in the low density lipoprotein based on the degree of color development of a coloring matter (see Japanese Laid-Open Patent Publication No. Hei 10-84997). This method reduces the reactivity of the enzyme with cholesterol included in lipoproteins other than the low-density lipoprotein in the presence of the amphoteric surface active agent.
The third proposed method treats the sample with polycation to oxidize only cholesterol in the low density lipoprotein with an enzyme and determines the concentration of cholesterol in the low density lipoprotein based on the degree of color development of a coloring matter (see U.S. Pat. No. 4,185,963).
The fourth proposed method uses porous silica to adsorb the high density lipoprotein, and causes polyanion/divalent cation to be bonded to the chylomicron and the low density lipoprotein and form an insoluble complex. The method then removes the complex as a precipitate from the solution, oxidizes only cholesterol in the low density lipoprotein with an enzyme, and determines the concentration of cholesterol in the low density lipoprotein based on the degree of color development of a coloring matter (see U.S. Pat. No. 5,401,466).
Any of the first through the fourth proposed methods discussed above enables cholesterol in the low density lipoprotein to be determined with high reproducibility and high accuracy, even in a sample having a high concentration of triglyceride.
All the first through the fourth methods determine the concentration of cholesterol based on the degree of color development of the coloring matter. These methods are thus not applicable for determination of cholesterol in originally colored samples like blood samples. For the determination of high reproducibility and high accuracy, the required process separately provides an enzyme-free first reagent and an enzyme-containing second reagent, adds only the first reagent to the sample first, and waits for addition of the second reagent until the reaction sufficiently proceeds. The procedures are accordingly rather complicated and troublesome.
The object of the present invention is thus to eliminate the above drawbacks and thus to provide a sensor that is applicable for measurement in blood sample and enables cholesterol in low density lipoprotein to be determined with high reproducibility and high accuracy by only one feed of a sample without requiring to separately add two or more reagents to the sample at different timings.
The object of the present invention is also to provide a method of determining cholesterol in the low density lipoprotein.
The present invention is directed to a cholesterol sensor for determining cholesterol in low density lipoprotein. The cholesterol sensor comprises: an electrically insulating base plate; an electrode system that is mounted on the base plate and includes at least a working electrode and a counter electrode; an enzyme layer formed on the base plate with the electrode system; and a reagent layer that is arranged before the enzyme layer in a sample solution supply path to the electrode system. The enzyme layer includes at least an oxidoreductase and an electron mediator. The reagent layer includes a reagent that attaches to lipoproteins other than the low density lipoprotein to form a water-soluble complex.
The present invention also provides a cholesterol sensor comprising: an electrically insulating base plate; an electrode system that is mounted on the base plate and includes at least a working electrode and a counter electrode; an enzyme layer formed on the base plate with the electrode system; a filter that is arranged before the enzyme layer in a sample solution supply path to the electrode system; and a reagent layer that is either carried on the filter or arranged before the filter in the sample solution supply path. The enzyme layer includes at least an oxidoreductase and an electron mediator. The reagent layer includes a reagent that functions to adsorb and/or agglutinate lipoproteins other than the low density lipoprotein.
The present invention further provides a method of determining cholesterol in low density lipoprotein. The method includes the steps of:
(a) causing a sample to react with any reagent or reagent group selected from the group consisting of (i), (ii), and (iii) specified below:
(i) xcex1-cyclodextrin sulfate, dextran sulfate, magnesium ion, and polyoxyethylene-polyoxypropylene block copolyether,
(ii) an amphoteric surface active agent and aliphatic amines having either of carboxyl group and sulfonate group, and
(iii) polycation;
(b) subsequently causing the sample to react with an oxidoreductase, which specifically reacts with cholesterol, and an electron mediator; and
(c) electrochemically oxidizing the electron mediator, which has been reduced in the step (b), so as to measure a value of oxidation current.
The present invention is also directed to a method of determining cholesterol in low density lipoprotein. The method includes the steps of:
(a) causing a sample to react with polyanion, divalent cation, and either an antibody against high density lipoprotein or silica;
(b) subsequently causing the sample to react with an oxidoreductase, which specifically reacts with cholesterol, and an electron mediator; and
(c) electrochemically oxidizing the electron mediator, which has been reduced in the step (b), so as to measure a value of oxidation current.